A variety of hormones and neurotransmitters regulate the biological functions through specific receptor proteins located in a cell membrane. Many of these receptor proteins are coupled with guanine nucleotide-binding proteins (hereinafter sometimes referred to as G proteins) and evoke the intracellular signal transduction via activation of the G proteins. These receptor proteins possess the common structure, i.e. seven transmembrane domains and are thus collectively referred to as G protein-coupled receptors or seven-transmembrane receptors (7TMR).
An important regulation of biological functions, such as homeostasis, reproduction, individual development, metabolism, growth, regulations of nervous system, respiratory system, digestive system and metabolic system, and sensory system is conducted through an interaction between these hormones or neurotransmitters and G protein-coupled receptor proteins. In this context, it is known that there are various receptor proteins for hormones and neurotransmitters for the regulation of biological functions and these proteins play an important role for regulating the functions. However, it is not much clear as to whether unknown active substances (e.g. hormones, neurotransmitters, etc.) and receptors thereof still exist.
In recent years, using the fact that G protein-coupled receptor proteins represent similarities in their partial amino acid sequences, the search for DNA encoding a novel receptor protein is conducted by Polymerase Chain Reaction (hereinafter abbreviated as PCR) method. Therefore, many orphan G protein-coupled receptor proteins whose ligand are not known, are cloned (Libert, F., et al. Science, 244, 569–572, 1989, Welch, S. K., et al., Biochem. Biophys. Res. Commun., 209, 609–613, 1995, Marchese, A., et al., Genomics, 23, 609–618, 1994, Marchese, A., Genomics, 29, 335–344, 1995). Novel G protein-coupled receptor proteins are also found by random analysis of genomic DNA or cDNA sequences (Nomura, N., et al., DNA Research vol. 1, 27–25, 1994). General methods for determining a ligand to an orphan G protein-coupled receptor protein are only to predict the ligand from similarity of the primary structure of G protein-coupled receptor protein. However, since many G protein-coupled receptor proteins represent low homology with the known receptors, it is difficult to predict a ligand only from the similarity of the primary structure unless the receptor protein is a subtype receptor for the known ligand. On the other hand, many orphan G protein-coupled receptor proteins are found by genetic analysis. So, it is estimated that there are many unknown ligands still remained. Nevertheless, only a few ligands for G protein-coupled receptor proteins are actually identified.
On the other hand, Neuromedin U is a peptide, which was isolated and purified from porcine spinal cords, with setting a rat uterus smooth muscle contraction activity as an index. Two kinds of Neuromedin U, Neuromedin U-8 having 8 amino acid residues and Neuromedin U-25 having 25 amino acid residues are first reported (Minamino, N. et al., Biochem. Biophys. Res. Commun. 130, 1078–1085, 1985). Since the sequence of Neuromedin U-8 is identical to C-terminal sequence of Neuromedin U-25 and the upstream region contains a basic amino acid pair often seen in the cleavage site for processing, both Neuromedin U are expected to be derived from a common precursor. Also, other physiological functions besides the smooth muscle contraction activity are widely known. Such functions reportedly include, for example, increase in blood pressure (Minamino. N. et al.), decrease in bloodstream of intestine (Sumi, S. et al., Life Sci. 41, 1585–1590, 1987), adjustment of ion transportation in intestine (Brown, D. R. and Quito, F. L., Eur. J. Pharmacol. 155, 159–162, 1998) and increase in ACTH and subsequent increase in corticosterone after hypodermic administration of Neuromedin U (Malendowicz, L. K. et al., In Vivo, 7, 419–422, 1993).